Project Summary Receptors from the ionotropic glutamate family are responsible for most of the fast excitatory neurotransmissions in the central nervous system. Within this family, the N-methyl-D-aspartate (NMDA) receptors? distinctive gating properties as well as their high calcium permeability make them a fundamental component of long-term potentiation and neuronal plasticity, vital in learning and memory formation. Both hyper- and hypo-activity of the receptors have been implicated in neurological disorders such as Alzheimer?s, schizophrenia, depression, and chronic pain, making NMDARs excellent candidates for the development of therapeutics. Understanding the molecular mechanism responsible for NMDAR activity modulation is essential in the development of specific therapeutics. The overall goal of this project is to determine the structural mechanisms underlying the channel opening and the zinc inhibition for the GluN1/GluN2A subtype of NMDARs. The GluN1/Glun2A subtype is expressed widely throughout the central nervous system and has a unique high-affinity binding site for zinc. Nanomolar concentrations of zinc inhibit the GluN1/GluN2A NMDAR, a characteristic which has recently been shown to have physiological relevance in pain hypersensitivity. While the structure of the isolated amino-terminal domain of the GluN1/GluN2A receptor in the zinc-bound or zinc- free states has been obtained, the structural justification for zinc modulation of the NMDA receptors has remained elusive. The first goal of this proposal is to understand the structural basis for the zinc inhibition of the GluN1/GluN2A NMDAR. In support of the first goal, I will obtain the structure of the receptor in the zinc- bound and zinc-chelated states using single particle cryo-EM. A second fundamental question in this field, and the second goal of this project, is the understanding of the mechanism of NMDA receptor activation and channel opening, requiring the structure of the receptor in open-pore conformation. Among subtypes of NMDARs the GluN1/GluN2A receptor has the highest probability of being in the open conformation when bound by agonists, making it an ideal choice for obtaining the open conformation of the receptor. In support of this goal, I will obtained the structure of the receptor in the open-channel conformation using a combination of agonists and zinc-chelating reagents, small-molecule positive allosteric modulators, potentiating ligands, and finally by the generation of state-selective peptide ligands. These peptide ligands will be generated and characterized using mRNA display coupled with high-throughput DNA sequencing. The structures obtained as the result of this proposal will elucidate the molecular underpinnings of the channel opening as well as the mechanism of high-affinity zinc-inhibition for the GluN1/GluN2A NMDAR, representing a significant advancement in the field. Furthermore, any peptide ligands generated by this work can also serve as starting points for the development of GluN1/GluN2A specific therapeutics.